Methods for treating tumors

ABSTRACT

The present invention provides methods for increasing the sensitivity of a tumour to immunotherapy, chemotherapy or radiotherapy by administering an effective amount of an oligonucleotide that inhibits the binding of miR-27a, or a variant thereof, to its target mRNA. Oligonucleotides used in the invention are typically in the form of a blockmirs used as an adjunctive therapy to inhibit tumour growth, normalise and/or improve function of tumour vasculature, and/or promote immune cell infiltration of tumours.

FIELD OF THE INVENTION

The present invention relates generally to the use of oligonucleotidesas an adjunctive therapy in inhibition of tumour growth, fornormalisation and/or improving function of tumour vasculature, and/orfor promoting immune cell infiltration of tumours.

BACKGROUND OF THE INVENTION

As a tumour grows its requirements for nutrients become greater andangiogenesis is induced to supply new vasculature to the tumour. Thistumour vasculature differs from normal vasculature, being poorlyorganised and having aberrant vessel walls. Fewer tight junctions, gapsbetween endothelial cells, loosely attached pericytes, and an abnormalbasement membrane and extracellular matrix result in a hyperpermeableendothelial barrier. This poor vascularisation contributes to a hypoxicmicroenvironment in the tumour which induces biochemical pathways thatpromote further tumour development, eg via vascular endothelial growthfactor (VEGF) and hypoxia inducible factor (HIF). As such, hypoxiapromotes angiogenesis, inflammation, cancer stem cell morphology,immunosuppression and anaerobic metabolism. The hypoxic environment canalso induce metastasis of the tumour.

A further consequence of poor perfusion of the tumour is an impairmentof therapeutic treatments, such as chemotherapy and immunotherapy, whichrely on the vasculature for delivery. Anti-angiogenic agents have beenused with some success to temporarily normalize tumour vasculature andalleviate hypoxia (see, for example, Jain, 2001 Nat. Med. 9, 685-693)and adjunctive therapy combining anti-angiogenic agents withchemotherapeutic agents has been shown to increase survival in somecancer patients. However anti-angiogenic agents can cause extensivedamage to, including destruction of, tumour vessels and vesselnormalisation does not persist. Thus current tumour treatments posedifficulties in their effective delivery, and high doses of therapeuticagents required in treatment regimens which may produce unwanted sideeffects.

Accordingly, there is a need for novel treatments that effectivelytarget the tumour vasculature without causing the damaging effects ofknown anti-angiogenic therapies.

SUMMARY OF THE INVENTION

Disclosed herein is the use of oligonucleotides for modulating tumourstroma, tumour vasculature, metastasis and sensitivity to treatment,wherein the oligonucleotides comprise sequences complementary to andcapable of binding to, the sequence shown in SEQ ID NO: 1.

In a first aspect the present invention provides a method for increasingthe sensitivity of a tumour to immunotherapy, chemotherapy orradiotherapy, wherein the method comprises administering to a subject inneed thereof an effective amount of an oligonucleotide comprising acontiguous sequence complementary to at least 8 contiguous bases of anRNA sequence comprising SEQ ID NO: 1, or SEQ ID NO: 1 comprising 1, 2 or3 substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof, or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.

The method may further comprise immunotherapy, chemotherapy orradiotherapy of said tumour in said subject. In an embodiment, theoligonucleotide is administered to the subject prior to, concomitantlywith, after, or otherwise in combination with, immunotherapy,chemotherapy or radiotherapy of said tumour.

In a particular embodiment, the oligonucleotide comprises a contiguoussequence complementary to at least 8 contiguous bases of an RNA sequencecomprising SEQ ID NO: 2, or SEQ ID NO: 2 comprising 1, 2 or 3substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.

Typically the miR-27a miRNA is hsa-miR-27a comprising the nucleotidesequence set forth in SEQ ID NO: 9.

In an embodiment, the oligonucleotide comprises a contiguous sequencecomplementary to a sequence of at least or about 7 bases, at least orabout 8 bases, at least or about 9 bases, at least or about 10 bases, atleast or about 11 bases, at least or about 12 bases, at least or about13 bases, at least or about 14 bases, at least or about 15 bases, atleast or about 16 bases, at least or about 17 bases, at least or about18 bases, at least or about 19 bases, at least or about 20 bases, atleast or about 22 bases, at least or about 25 bases, at least or about30 bases, or at least or about 35 bases of SEQ ID NO: 2, or SEQ ID NO: 2comprising 1, 2 or 3 substitutions.

Typically the oligonucleotide binds to positions 22-27 of SEQ ID NO: 2.

In a particular embodiment, base pairing between the oligonucleotide andSEQ ID NO: 2 includes positions 8-28, 8-27, 9-27, 10-27, 11-27, 12-27,13-27, 14-27, 15-27, 16-27, 17-27, 18-27, 19-27, 20-27, 21-27, 9-28,10-28, 11-28, 12-28, 13-28, 14-28, 15-28, 16-28, 17-28, 18-28, 19-28,20-28 or 21-28 of SEQ ID NO: 2.

In a further particular embodiment, the oligonucleotide comprises thesequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

In a further particular embodiment, the oligonucleotide comprises one ormore modified nucleobases. In exemplary embodiments, the modifiednucleobase may be selected from an LNA nucleobase, a UNA nucleobase anda 2′ O-methyl nucleobase.

In a further particular embodiment, the oligonucleotide comprises asequence set forth in SEQ ID NO: 5.

In a further particular embodiment, the immunotherapy comprises immunestimulation, comprising adoptive cell transfer or the administration ofone or more anti-tumour or immune checkpoint antibodies, anti-tumourvaccines or other immune cell modulating agents. In an embodiment,adoptive cell transfer comprises the transfer of autologous tumourinfiltrating lymphocytes. In a further embodiment, the anti-tumourantibodies comprise anti-PD-1 antibodies.

In a second aspect the present invention provides a method formodulating tumour metastasis, the method comprising exposing a tumour toan effective amount of an oligonucleotide comprising a contiguoussequence complementary to at least 8 contiguous bases of an RNA sequencecomprising SEQ ID NO: 1, or SEQ ID NO: 1 comprising 1, 2 or 3substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.

Typically, modulating tumour metastasis comprises reducing tumourmetastasis.

In a third aspect the present invention provides a method fornormalising tumour vasculature and/or improving vascular function in atumour, the method comprising exposing a tumour to an effective amountof an oligonucleotide comprising a contiguous sequence complementary toat least 8 contiguous bases of an RNA sequence comprising SEQ ID NO: 1,or SEQ ID NO: 1 comprising 1, 2 or 3 substitutions, wherein theoligonucleotide inhibits the binding of miR-27a, a variant thereof or amiRNA comprising a seed region comprising the sequence UCACAG, to saidRNA.

In a particular embodiment, normalising the tumour vasculature and/orimproving vessel function comprises or is characterized by one or moreof: change in morphology of endothelial cells, change in VE-cadherinexpression, selective loss of large vessels; increase in number of smallvessels, increased pericyte coverage of vessels, altered collagen IVcoverage of vessels, reduced vessel permeability, reduced vesselhypoxia, increased vessel perfusion, and enhanced infiltration of immunecells. In a particular embodiment the immune cells are lymphocytes. In afurther embodiment the lymphocytes comprise CD8+ T cells, CD4+ T cellsand/or NK cells.

In a fourth aspect the present invention provides a method for reducingtumour hypoxia, the method comprising exposing a tumour to an effectiveamount of an oligonucleotide comprising a contiguous sequencecomplementary to at least 8 contiguous bases of an RNA sequencecomprising SEQ ID NO: 1, or SEQ ID NO: 1 comprising 1, 2 or 3substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.

In a fifth aspect the present invention provides a method for increasingcell death of tumour cells, the method comprising exposing a tumour toan effective amount of an oligonucleotide comprising a contiguoussequence complementary to at least 8 contiguous bases of an RNA sequencecomprising SEQ ID NO: 1, or SEQ ID NO: 1 comprising 1, 2 or 3substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.

In a sixth aspect the present invention provides use of anoligonucleotide comprising a contiguous sequence complementary to atleast 8 contiguous bases of an RNA sequence comprising SEQ ID NO: 1, orSEQ ID NO: 1 comprising 1, 2 or 3 substitutions, wherein theoligonucleotide inhibits the binding of miR-27a, a variant thereof or amiRNA comprising a seed region comprising the sequence UCACAG, to saidRNA for the manufacture of a medicament for sensitising tumours,modulating tumour metastasis, normalising tumour vasculature and/orpromoting cell death of tumour cells.

In particular embodiments of the above aspects, the tumour is a solidtumour.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are described herein, by way ofnon-limiting example only, with reference to the following figures.

FIG. 1. Blockmir CD5-2 facilitates the infiltration of adoptivelytransferred CD8+ T cells into RIP-TAG tumours. (A) Representativecomposite images of tumours. DAPI (blue, nuclei) and CD8 (red, cytotoxicT cells) given either control Blockmir or Blockmir CD5-2 (2 differentimages are shown). (B) Quantification of the ratio of infiltrated CD8 Tcells to cells in the visual field. Data represents mean±SEM. n=3 miceper group, paired t-test. (C) Quantification of the ratio of infiltratedCD8 T cells to cells in the visual field when removing one mouse of 3that received the control Blockmir, since it showed an unusually highinfiltration (>1 SD away from the mean). Data represents mean±SEM. n=2mice treated with control and 3 mice treated with Blockmir CD5-2.

FIG. 2. Effect of Blockmir CD5-2 on the infiltration of endogenous CD8+cytotoxic T cell into the B16F10 melanoma tumours. (A) Quantification ofthe number of CD8+ cells per field (two fields per mouse). Datarepresents mean±SEM. n=5 mice per group, paired t-test. (B)Representative composite images of B16F10 tumours. DAPI (blue, nuclei),CD8 (white, cytotoxic T cells) and CD31 (red, endothelium). The imagesshow more T cells infiltrate into the middle of the tumour parenchyma inthe Blockmir CD5-2 treated mice (right panel) compared to that in thecontrol treated mice (left panel). The label miRNA refers to BlockmirCD5-2. The distance between leading edge of invasive CD8+ T cells andedge of tumour section was quantified. Data represents mean±SEM. **,P<0.01, n=5 mice per group, two independent experiments, paired t-test.(C) Representative confocal images of B16F10 melanoma sections stainedfor TUNEL (green) to visualize the apoptotic cells. The percentage ofTUNEL positive cells was quantified. Data represents mean±SEM. n=6 miceper group, paired t-test. (D) Growth curve of primary B16F10 tumoursgiven 30 mg/kg of control or CD5-2 intravenously into nude mice. Datarepresents mean±SEM, 7 mice from three independent experiments, pairedt-test. (E) Representative images of pericyte coverage in nude micetreated with control or CD5-2. Pericyte coverage was quantified aspreviously. Data represents mean±SEM. *, P<0.05, n=3 mice per group,three independent experiments, paired t-test. (F) Representative imagesof smooth muscle cell coverage in nude mice treated with control orCD5-2. Smooth muscle cell coverage was quantified as previously. Datarepresents mean±SEM. *, P<0.05, n=3 mice per group, three independentexperiments, paired t-test.

FIG. 3. Blockmir CD5-2 reduces average tumour vessel volume. (A)Representative confocal images of B16F10 melanoma sections (day 5following the injection of control or CD5-2) stained for CD31 (red) tovisualize tumour vessels in the treatment of control or Blockmir CD5-2,at 6-8 days of growth. (B) Quantification of average vessel volume. Datarepresents mean±SD. **, P<0.01, n=5 mice per group, paired t-test. (C)Quantification of the number of vessels per field. Data representsmean±SD. *, P<0.05, n=5 mice per group, paired t-test. 50 μm sectionswere taken for the analysis.

FIG. 4. SEM micrographs of tumour vessels in the treatment of control orBlockmir CD5-2. Left panel: mice were given control Blockmir. Abnormaltumour vessel containing multilayers of disconnected endothelial cellswith luminal protrusions in tumour. Right panel: mice were givenBlockmir CD5-2. Vessel lined by monolayer of cobblestone endothelialcells. B16F10 tumours at Day 12. Representative of 4 vessels imaged forcontrol and 4 for CD5-2 treated mice.

FIG. 5. Blockmir CD5-2 induces pericyte and smooth muscle cell coveragein B16F10 melanoma model. (A) Endothelium and associated pericytes werevisualised by CD31 (red) and NG2 (green) immunofluorescence stainingrespectively of B16F10 tumours from control or Blockmir CD5-2 treatedmice. Bottom row, high magnification of selected area in top row. (B)Pericyte coverage was quantified by calculating the percent fraction ofvessel length that overlapped with NG2 staining in the image of thevessels to determine direct contact between the two cell types. Datarepresents mean±SEM. *, P<0.05, n=8 mice per group, paired t-test. (C)Endothelium and associated smooth muscle cells were visualised by CD31(red) or αSMC (green) immunofluorescence staining respectively of B16F10tumour implants. Bottom row, high magnification of selected area in toprow. (D) Smooth muscle cell coverage was quantified by calculating thepercent fraction of vessel length that overlapped with αSMC staining inthe image of the vessels to determine direct contact between the twocell types. Data represents mean±SEM. *, P<0.05, n=8 mice per group,paired t-test.

FIG. 6. Effects of Blockmir CD5-2 on VE-Cadherin expression in B16F10melanoma tumour vessels. (A) Representative images of VE-Cadherinexpression (green) in tumour vessels stained for CD31 (red). Topimage=control Blockmir, bottom image representative image of BlockmirCD5-2 treated tumours. (B) The ratio of the fluorescence intensity ofVE-Cadherin to CD31 was determined and is presented as relative values.Data represents mean±SD. n=5 mice per group. *, P<0.05, paired t-test.

FIG. 7. Blockmir CD5-2 promotes basement membrane support in the B16F10melanoma model. (A) Endothelium and associated basement membrane werevisualised by CD31 (red) and Collagen IV (green) immunofluorescencestaining respectively of B16F10 tumours from mice treated with controlor Blockmir CD5-2. (B) Basement membrane support of tumour vasculaturewas quantified by calculating the percent fraction of vessel length thatoverlapped with Collagen IV staining in the image of the vessels todetermine direct contact between endothelial cells and basementmembrane. Data represents mean±SD. **, P<0.01, n=5 mice per group,paired t-test.

FIG. 8. Blockmir CD5-2 decreases tumour vascular permeability in B16F10melanoma model. (A) Representative images of tumour vessel leakiness inthe tumours from mice treated with control or Blockmir CD5-2. R50fluorescent microspheres were injected intravenously into C57BL/6 micebearing B16F10 tumours. The extravasated 50 nm fluorescent microspheres(green) from tumour vessels stained for CD31 (red) are shown. (B) Toquantify the level of microsphere leakage and standardize it for tumourvessel area, the ratio of the number of microspheres to CD31 area wasdetermined and is presented as relative values. Data represents mean±SD.**, P<0.01, n=6 mice per group, paired t-test. (C) Representative imagesof fibrinogen deposition in the treatment of control or CD5-2 are shown.(D) To quantify the level of fibrinogen deposition and standardize itfor tumour vessel area, the ratio of the area of fibrinogen to vesselwas determined and is presented as relative values. Data representsmean±SEM. *, P<0.05, n=6 mice per group, paired t-test.

FIG. 9. Blockmir CD5-2 promotes tumour vascular perfusion in B16F10melanoma model. (A) Representative images of tumour vascular perfusionin mice treated with control or Blockmir CD5-2. FITC-conjugated lectinwas injected intravenously into C57BL/6 mice bearing B16F10 tumours.Double positive staining for FITC-conjugated lectin (green) and CD31(red) was used to evaluate the perfused tumour vessels. (B) To quantifythe percentage of perfused vessels (yellow), the ratio of the number ofperfused vessels to total vessels was determined and is presented asrelative values. Data represents mean±SD. *, P<0.05, n=6 mice per group,paired t-test.

FIG. 10. Blockmir CD5-2 diminishes tumour hypoxia in the B16F10 melanomamodel. (A) Representative images of tumour hypoxia in the tumours frommice treated with control or Blockmir CD5-2. Hypoxia probe Hypoxyprobe-1was injected intravenously into C57BL/6 mice bearing B16F10 tumours.Double positive staining for pimonidazole (green) and CD31 (red) wasused to evaluate the level of tumour hypoxia. (B) Quantification oftumour hypoxic area (green) in the presence of control or BlockmirCD5-2. Data represents mean±SD. *, P<0.05, n=6 mice per group, pairedt-test.

FIG. 11. Blockmir CD5-2 promotes vascular perfusion in RIP-TAG5 tumourmodel. (A) Representative images of tumour vascular perfusion in thetreatment of control (Ctrl) or Blockmir CD5-2 (miRNA). FITC-conjugatedlectin was injected intravenously into 27-week old RIP-TAG5 mice. Doublepositive staining for FITC-conjugated lectin (green) and CD31 (red) wasused to evaluate the perfused vessels. (B) To quantify the percentage ofperfused vessels (yellow), the ratio of the number of perfused vesselsto total vessels was determined and is presented as relative values.Data represents mean±SEM. *, P<0.05, n=7 mice treated with control and 3mice treated with Blockmir CD5-2, unpaired t-test.

FIG. 12. CD5-2 enhances immunotherapeutic effects. (A) Representativeimages of tumour vascular perfusion in RIP-Tag5 pancreatic tumours.Double positive staining for FITC-conjugated lectin (green) and CD31(red) was used to evaluate the perfused tumour vessels. To quantify thepercentage of perfused vessels (yellow), the ratio of the number ofperfused vessels to total vessels was determined and is presented asrelative values. Data represents mean±SEM. *, P<0.05, n=3-5 mice pergroup, unpaired t-test. (B) Adoptive transfer of CD8+ T cells inRIP-Tag5 pancreatic tumours. CD8+ surface area (%) was quantified. Datarepresents mean±SEM. *, P<0.05, n=3-5 mice per group, unpaired t-test.(C) Growth curve of MC38 tumours. Anti-PD-1 or control IgG given IP onday 7, 11, 14. Control or CD5-2 was given i.v. on day 7. Data representsmean±SEM. ***, P<0.001; **, P<0.01; *, P<0.05, n=8 mice per group,two-way ANOVA test. (D) Growth curve of MC38 tumours. Anti-PD-1 orcontrol IgG given i.p. on day 7, 11, 14. Control or CD5-2 was given i.v.on day 6, 9, 12. Data represents mean±SEM. ***, P<0.001; **, P<0.01; *,P<0.05, n=8 mice per group, two-way ANOVA test. (E) CD8/Gr1 hi ratio ofMC38 tumours with single injection of CD5-2. Data represents mean±SEM.*, P<0.05; **, P<0.01, n=7-8 mice per group, unpaired t-test. (F) Thepercentage of CD8+ T cells that are Granzyme B positive. Data representsmean±SEM. *, P<0.05; **, P<0.01, n=7-8 mice per group, unpaired t-test.

FIG. 13. Effects of Blockmir CD5-2 on metastasis of B16F10 melanoma.2×10⁵ B16F10-luciferase cells were injected via tail vein into albinoC57BL/6 mice. Control or Blockmir CD5-2 was intravenously injected intomice two days before and two days after the cell injection. (A)Bioluminescent imaging of metastasis 12 days following the cellinjection. (B) Bioluminescent imaging of metastasis 15 days followingthe cell injection. (C) Bioluminescent imaging of metastasis 18 daysfollowing the cell injection. (D) Quantification of bioluminescence onday 18. Data represents mean±SD, 3 experiments.

FIG. 14. Bioluminescent images of lung and liver metastasis. Lungs andlivers of mice injected with B16F10 melanoma and treated with control orBlockmir CD5-2 and harvested 18 days following the cell injection.

The subject specification contains amino acid and nucleotide sequenceinformation prepared using the programme PatentIn Version 3.4, presentedherein in a Sequence Listing. Nucleotide and amino acid sequences arereferred to by a sequence identifier number (SEQ ID NO:). The SEQ IDNOs: correspond numerically to the sequence identifiers <400>1 (SEQ IDNO:1), <400>2 (SEQ ID NO:2), etc. Specifically, the miR-27a target‘anti-seed’ region in the 3′UTR of VE-cadherin (CDH5) is shown in SEQ IDNO: 1. The region of the 3′UTR of human VE-cadherin containing themiR-27a ‘anti-seed’ region is shown in SEQ ID NO: 2. SEQ ID NOs: 3 to 8show the sequences of exemplary oligonucleotides. The mature sequence ofthe human miR-27a (hsa_miR-27a) is shown in SEQ ID NO: 9.

DETAILED DESCRIPTION

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

In the context of this specification, the term “about” is understood torefer to a range of numbers that a person of skill in the art wouldconsider equivalent to the recited value in the context of achieving thesame function or result.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

As used herein the term “oligonucleotide” refers to a single-strandedsequence of ribonucleotide or deoxyribonucleotide bases, known analoguesof natural nucleotides, or mixtures thereof. An “oligonucleotide”comprises a nucleic-acid based molecule including DNA, RNA, PNA, LNA,UNA or any combination thereof. An oligonucleotide that predominantlycomprises ribonucleotide bases, natural or non-natural, may be referredto as an RNA oligonucleotide. Oligonucleotides are typically short (forexample less than 50 nucleotides in length) sequences that may beprepared by any suitable method, including, for example, direct chemicalsynthesis or cloning and restriction of appropriate sequences.

“Antisense oligonucleotides” are oligonucleotides complementary to aspecific DNA or RNA sequence. Typically in the context of the presentinvention an antisense oligonucleotide is an RNA oligonucleotidecomplementary to a specific mRNA or miRNA. The antisense oligonucleotidebinds to and silences or represses, partially of fully, the activity ofits complementary miRNA. Not all bases in an antisense oligonucleotideneed be complementary to the ‘target’ or miRNA sequence; theoligonucleotide need only contain sufficient complementary bases toenable the oligonucleotide to recognise the target. An oligonucleotidemay also include additional bases. The antisense oligonucleotidesequence may be an unmodified ribonucleotide sequence or may bechemically modified or conjugated by a variety of means as describedherein.

The term “polynucleotide” as used herein refers to a single- ordouble-stranded polymer of deoxyribonucleotide, ribonucleotide bases orknown analogues of natural nucleotides, or mixtures thereof. A“polynucleotide” comprises a nucleic-acid based molecule including DNA,RNA, PNA, LNA, UNA or any combination thereof. The term includesreference to the specified sequence as well as to the sequencecomplimentary thereto, unless otherwise indicated. Polynucleotides maybe chemically modified by a variety of means known to those skilled inthe art. Thus a “polynucleotide” comprises a nucleic-acid based moleculeincluding DNA, RNA, PNA, LNA, UNA or any combination thereof.

As used herein in relation to oligonucleotides and polynucleotides, theterm “nucleotide” refers to a single nucleobase or monomer unit withinthe oligonucleotide or polynucleotide. The terms “nucleotide” and“monomer” may be used interchangeably herein. The nucleobase may be partof a DNA, RNA, INA, LNA, UNA or combination of any two or more thereof)oligonucleotide or polynucleotide. In some embodiments, the nucleobasemay be a universal base. Modified nucleobases are also contemplated bythe present invention, as described hereinbelow.

The term “variant” as used herein refers to substantially similarsequences. Generally, polypeptide sequence variants also possessqualitative biological activity in common, such as receptor bindingactivity. Further, these polypeptide sequence variants may share atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity. The term “sequence identityor “percentage of sequence identity” may be determined by comparing twooptimally aligned sequences or subsequences over a comparison window orspan, wherein the portion of the polynucleotide sequence in thecomparison window may optionally comprise additions or deletions (i.e.,gaps) as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.

The term “complementary” as used herein refers to the ability of twosingle-stranded nucleotide sequences to base pair, typically accordingto the Watson-Crick base pairing rules, that is, between G and C andbetween A and T or U. In some embodiments, G also pairs to U and viceversa to form a so-called wobble base pair. In another embodiment, thebase inosine (I) may be included within an oligonucleotide of theinvention. I base pairs to A, C and U. In still another embodiment,universal bases may be used. Universal bases can typically base pair toG, C, A, U and T. Often universal bases do not form hydrogen bonds withthe opposing base on the other strand. In still another embodiment, acomplementary sequence refers to a contiguous sequence exclusively ofWatson-Crick base pairs. For two nucleotide molecules to becomplementary they need not display 100% complementarity across the basepairing regions, but rather there must be sufficient complementarity toenable base pairing to occur. Thus a degree of mismatching between thesequences may be tolerated and the sequences may still be complementary.As used herein, the term “capable of base pairing with” is usedinterchangeably with “complementary to”.

The term “substitution” as used herein refers to a nucleobase at aparticular position within an oligonucleotide or polynucleotide havingbeen substituted for another nucleobase. The substitution may be, forexample, because of the presence of a single nucleotide polymorphism inthe target RNA. The term substitution also encompasses deletions ofnucleobases and additions of nucleobases.

The term “Blockmir” as used herein refers to a steric blockingoligonucleotide that binds to an RNA target blocking the ability of oneor more miRNA species from binding to, and affecting the activity of,said target. Blockmirs are constructed so as to be incapable ofrecruiting cellular RNAi machinery or RNase H. RNAi machinery refers tothe cellular components necessary for the activity of siRNAs and miRNAsor for the RNAi pathway. A major component of the RNAi machinery is theRNA induced silencing complex (the RISC complex). Blockmirs aredescribed, for example, in WO 2008/061537, WO 2012/069059 and WO2014/053014, the disclosures of which are incorporated herein byreference.

In the context of this specification, the term “activity” as it pertainsto a polynucleotide (e.g. a DNA, mRNA or miRNA), protein or polypeptidemeans any one or more cellular function, action, effect or influenceexerted by the polynucleotide, protein or polypeptide. For example, inthe context of a mRNA, activity will typically refer to expression ofthe mRNA, i.e. translation into a protein or peptide. Thus, regulationof the activity of a target mRNA by an oligonucleotide as describedherein may include degradation of the mRNA and/or translationalregulation. Regulation of mRNA activity may also include affectingintracellular transport of the mRNA.

The term “inhibiting” and variations thereof such as “inhibition” and“inhibits” as used herein do not necessarily imply the completeinhibition of the specified event, activity or function. Rather, theinhibition may be to an extent, and/or for a time, sufficient to producethe desired effect. Inhibition may be prevention, retardation, reductionor otherwise hindrance of the event, activity or function. Suchinhibition may be in magnitude and/or be temporal in nature. Inparticular contexts, the terms “inhibit” and “prevent”, and variationsthereof may be used interchangeably.

The terms “promoting” and “inducing”, and variations thereof such as“promotion” and “inducement”, as used herein do not necessarily implythe complete promotion or inducement of the specified event, activity orfunction. Rather, the promotion or inducement may be to an extent,and/or for a time, sufficient to produce the desired effect. Thepromotion or inducement of angiogenesis by oligonucleotides of theinvention may be direct or indirect and may be in magnitude and/or betemporal in nature.

As used herein the term “effective amount” includes within its meaning anon-toxic but sufficient amount or dose of an agent or compound toprovide the desired effect. The exact amount or dose required will varyfrom subject to subject depending on factors such as the species beingtreated, the age and general condition of the subject, the severity ofthe condition being treated, the particular agent being administered andthe mode of administration and so forth. Thus, it is not possible tospecify an exact “effective amount”. However, for any given case, anappropriate “effective amount” may be determined by one of ordinaryskill in the art using only routine experimentation.

As used herein the terms “treating”, “treatment”, “preventing” and“prevention” refer to any and all uses which remedy a condition orsymptoms, prevent the establishment of a condition or disease, orotherwise prevent, hinder, retard, or reverse the progression of acondition or disease or other undesirable symptoms in any waywhatsoever. Thus the terms “treating” and “preventing” and the like areto be considered in their broadest context. For example, treatment doesnot necessarily imply that a patient is treated until total recovery. Inconditions which display or a characterized by multiple symptoms, thetreatment or prevention need not necessarily remedy, prevent, hinder,retard, or reverse all of said symptoms, but may prevent, hinder,retard, or reverse one or more of said symptoms. In the context of somedisorders, methods of the present invention involve “treating” thedisorder in terms of reducing or ameliorating the occurrence of a highlyundesirable event associated with the disorder or an irreversibleoutcome of the progression of the disorder but may not of itself preventthe initial occurrence of the event or outcome. Accordingly, treatmentincludes amelioration of the symptoms of a particular disorder orpreventing or otherwise reducing the risk of developing a particulardisorder.

As used herein the term “sensitivity” is used in its broadest context torefer to the ability of a cell to survive exposure to an agent designedto inhibit the growth of the cell, kill the cell or inhibit one or morecellular functions.

The term “subject” as used herein refers to mammals and includes humans,primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys),laboratory test animals (eg. mice, rabbits, rats, guinea pigs),companion animals (eg. dogs, cats) and captive wild animals (eg. foxes,kangaroos, deer). Preferably, the mammal is human or a laboratory testanimal. Even more preferably, the mammal is a human.

As described and exemplified herein the inventors have identifiedmethods for sensitising tumours to therapy and for the modulation oftumour metastasis and/or vasculature, comprising administration of anoligonucleotide capable of binding to the sequence CUGUGA blocking theability of a miRNA (such as miRNA miR-27a) to bind to said sequence andthereby inhibiting the miRNA from affecting the activity or expressionof a polynucleotide comprising said sequence. Typically the sequence ispresent in the 3′UTR of the VE-cadherin mRNA. In particular embodimentsadministration of the oligonucleotide sensitises the tumour toimmunotherapeutic, chemotherapeutic or radiotherapeutic treatments.

One aspect of the present invention provides a method for increasing thesensitivity of a tumour to immunotherapy, chemotherapy or radiotherapy,wherein the method comprises administration to a subject in need thereofof an effective amount of an oligonucleotide comprising a contiguoussequence complementary to at least 8 contiguous bases of an RNA sequencecomprising SEQ ID NO: 1, or SEQ ID NO: 1 comprising 1, 2 or 3substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.

Other aspects of the invention provide methods for modulating tumourmetastasis, for normalizing tumour vasculature, for normalizing vesselfunction in a tumour and/or for promoting cell death of cells in atumour, wherein the methods comprise exposing a tumour to an effectiveamount of an oligonucleotide comprising a contiguous sequencecomplementary to at least 8 contiguous bases of an RNA sequencecomprising SEQ ID NO: 1, or SEQ ID NO: 1 comprising 1, 2 or 3substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.

By way of example, normalising the tumour vasculature and/or improvingvessel function may comprise or be characterized by one or more of: achange in morphology of endothelial cells, change in VE-cadherinexpression, selective loss of large vessels; increase in number of smallvessels, increased pericyte coverage of vessels, altered collagen IVcoverage of vessels, reduced vessel permeability, reduced vesselhypoxia, increased vessel perfusion, and enhanced infiltration of immunecells, typically lymphocytes, including CD8+ T cells, CD4+ T cellsand/or NK cells.

Typically, cell death can occur by programmed cell death, such as byapoptosis and can include cell changes such as membrane blebbing, cellshrinkage, nuclear and/or DNA fragmentation and condensation ofchromatin.

Oligonucleotides

Typically, the miRNA comprising the seed sequence UCACAG is miR-27a. Thenucleotide sequence of mature human miR-27a (hsa-miR-27a) is provided inSEQ ID NO: 9. Additional sequence information for the miR-27a miRNA canbe found at http://microrna.sanqer.ac.uk/sequences/index.shtml. Alsocontemplated herein are variants of this miRNA. Variants includenucleotide sequences that are substantially similar to the sequence ofmiR-27a. For example, a variant miRNA may comprise a sequence displayingat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to SEQ ID NO:9.

Oligonucleotides for use in accordance with the present inventiontypically comprise a contiguous sequence complementary to a sequenceselected from the group consisting of at least about 9 contiguous bases,at least about 10 contiguous bases, at least about 11 contiguous bases,at least about 12 contiguous bases, at least about 13 contiguous bases,at least about 14 contiguous bases, at least about 15 contiguous bases,at least about 16 contiguous bases, at least about 17 contiguous bases,at least about 18 contiguous bases, at least about 19 contiguous bases,at least about 20 contiguous bases, at least about 22 contiguous bases,at least about 25 contiguous bases, at least about 30 contiguous bases,and at least about 35 contiguous bases of the sequence set forth in SEQID NO: 2 or the sequence of SEQ ID NO: 2 comprising 1, 2 or 3substitutions.

In an embodiment, the oligonucleotide may comprise a contiguous sequencecomplementary to a sequence selected from the group consisting of nomore than 8 contiguous bases, no more than 9 contiguous bases, no morethan 10 contiguous bases, no more than 11 contiguous bases, no more than12 contiguous bases, no more than 13 contiguous bases, no more than 14contiguous bases, no more than 15 contiguous bases, no more than 16contiguous bases, no more than 17 contiguous bases, no more than 18contiguous bases, no more than 19 contiguous bases, no more than 20contiguous bases, no more than 22 contiguous bases, no more than 25contiguous bases, no more than 30 contiguous bases, and no more than 35contiguous bases of the sequence set forth in SEQ ID NO: 2 or thesequence of SEQ ID NO: 2 comprising 1, 2 or 3 substitutions.

In another embodiment, the oligonucleotide may comprise a contiguoussequence complementary to a sequence selected from the group consistingof 8 contiguous bases, 9 contiguous bases, 10 contiguous bases, 11contiguous bases, 12 contiguous bases, 13 contiguous bases, 14contiguous bases, 15 contiguous bases, 16 contiguous bases, 17contiguous bases, 18 contiguous bases, 19 contiguous bases, 20contiguous bases, 21 contiguous bases, 22 contiguous bases, 23contiguous bases, 24 contiguous bases, 25 contiguous bases, 30contiguous bases, and 35 contiguous bases of the sequence set forth inSEQ ID NO: 2 or the sequence of SEQ ID NO: 2 comprising 1, 2 or 3substitutions.

Typically the oligonucleotide binds to positions 22-27 of SEQ ID NO: 2,this region representing the complement of the seed sequence of miR-27a,being the target site for miR-27a binding to the 3′UTR of theVE-cadherin mRNA (the ‘anti-seed’ region). Base pairing between theoligonucleotide and SEQ ID NO: 2 may include positions 8-28, 8-27, 9-27,10-27, 11-27, 12-27, 13-27, 14-27, 15-27, 16-27, 17-27, 18-27, 19-27,20-27, 21-27, 9-28, 10-28, 11-28, 12-28, 13-28, 14-28, 15-28, 16-28,17-28, 18-28, 19-28, 20-28 or 21-28 of SEQ ID NO: 2.

In one embodiment, base pairing between the oligonucleotide and thesequence of SEQ ID NO: 2 ends at position 27 of SEQ ID NO: 2. In otherembodiments, base pairing may end at position 28, 29, 30, 31, 32 or 33of SEQ ID NO: 2. In another embodiment, base pairing between theoligonucleotide and the sequence of SEQ ID NO: 2 begins at position 22of SEQ ID NO: 2. In other embodiments, base pairing may start atposition 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5of SEQ ID NO: 2.

Those skilled in the art will appreciate that oligonucleotides for usein accordance with the invention may be of any suitable length dependingon the precise function or use of the oligonucleotide. Typically, theoligonucleotides are between 8 and 25 bases in lengths. Even moretypically, the oligonucleotides are between 10 and 20 bases in length.

For strong binding to its target RNA, the length of the oligonucleotidemay be increased. In some cases, delivery into cells may be improved mayusing shorter oligonucleotides. Further, in other cases, the position ofthe oligonucleotide respective to the anti-seed sequence of the targetRNA may be adjusted. For example, the position of bases complementary toposition 22-27 of the target RNA of SEQ ID NO: 2 may be adjusted suchthat they are placed for example at the 5′end of the oligonucleotide, atthe 3′end of the oligonucleotide or in or towards the middle of theoligonucleotide. Typically, the position of bases complementary topositions 22-27 are placed in the oligonucleotide such that they startat position 1, position 2, position 3, position 4, position 5 orposition 6, or at a position upstream of position 2, position 3,position 4, position 5 or position 6 or at a position downstream ofposition 1, position 2, position 3, position 4, position 5 or position6, wherein the positions are counted from the 5′end of theoligonucleotide.

In some embodiments, the target RNA sequence, for example the sequenceof SEQ ID NO: 2 may comprise 1, 2 or 3 substitutions. Alternatively, thesequence may comprise no substitutions. Where substitutions are present,these may be located in the region of complementarity between theoligonucleotide and the target RNA. Substitutions may be singlenucleotide polymorphisms (SNPs) that may enhance or decrease miRNAregulation of the given target RNA. An SNP may create a new miRNA targetsite so as to cause aberrant miRNA regulation of the given target RNA.RNA editing may also give rise to substitutions.

Oligonucleotides for use in accordance with the invention may be capableof activating RNase H. RNase H cleaves the RNA part of a RNA-DNA duplexand the structural requirements for RNase H activation are well-known tothe skilled addressee. Similarly, oligonucleotides of the invention maybe capable of recruiting the cellular RNAi machinery and directing theRNAi machinery to the target RNA. This may result in cleavage of thetarget RNA or translational repression of the target RNA.

However in particular embodiments of the present invention, theoligonucleotides can neither recruit the RNAi machinery nor RNase H.Thus typically, oligonucleotides of the invention are capable ofblocking the activity of the RNAi machinery at a particular target RNA.The oligonucleotides may do so by sequestering the target sequence (themiRNA binding site) of the target RNA, such that the RNAi machinery willnot recognize the target sequence. Oligonucleotides of the inventionwith this activity may also be referred to as Blockmirs, because theyblock the regulatory activity of a given miRNA at a particular miRNAbinding site in target RNA. To achieve the ability to preventrecruitment or activation of RNase H by oligonucleotides of theinvention, the oligonucleotides typically do not comprise 5 or morecontiguous DNA nucleobases.

The oligonucleotides for use in accordance with the invention maycomprise a variety of sequence and structural modifications, dependingon the use and function of the oligonucleotide, as will be describedfurther below. Those skilled in the art will appreciate that thesequence and structural modifications described herein are exemplaryonly, and the scope of the present invention should not be limited byreference to those modifications, but rather additional modificationsknown to those skilled in the art may also be employed provided theoligonucleotide retains the desired function or activity.

By way of example only, the oligonucleotide sequence may be modified bythe addition of one or more phosphorothioate (for examplephosphoromonothioate or phosphorodithioate) linkages between residues inthe sequence, or the inclusion of one or morpholine rings into thebackbone. Alternative non-phosphate linkages between residues includephosphonate, hydroxlamine, hydroxylhydrazinyl, amide and carbamatelinkages, methylphosphonates, phosphorothiolates, phosphoramidates orboron derivatives. The nucleotide residues present in theoligonucleotide may be naturally occurring nucleotides or may bemodified nucleotides. Suitable modified nucleotides include 2′-O-methylnucleotides, 2′-O-flouro nucleotides, 2′-O-methoxyethyl nucleotides,universal nucleobases such as 5-nitro-indole; LNA, UNA, PNA and INAnucleobases, 2′-deoxy-2′-fluoro-arabinonucleic acid (FANA) andarabinonucleic acid (ANA). The ribose sugar moiety that occurs naturallyin ribonucleosides may be replaced, for example with a hexose sugar,polycyclic heteroalkyl ring, or cyclohexenyl group. Alternatively, or inaddition, the oligonucleotide sequence may be conjugated to one or moresuitable chemical moieties at one or both ends. For example, theoligonucleotide may be conjugated to cholesterol via a suitable linkagesuch as a hydroxyprolinol linkage at the 3′ end. As a further example,the oligonucleotide may be conjugated to N-acetylgalactosamine (GalNAc).

Particular modifications of interest include those that increase theaffinity of the oligonucleotide for complementary sequences, i.e.increase the melting temperature of the oligonucleotide base paired to acomplementary sequence, or increase the biostability of theoligonucleotide. Such modifications include 2′-O-flouro, 2′-O-methyl,2′-O-methoxyethyl groups. The use of LNA, UNA, PNA and INA monomers arealso typically employed. For shorter oligonucleotides, typically ahigher percentage of affinity increasing modifications are present. Ifthe oligonucleotide is less than 12 or 10 nucleobases in length, it maybe composed entirely of affinity increasing units, e.g. LNA monomers,UNA monomers or 2′-O-methyl RNA nucleobases.

In particular embodiments, the fraction of monomers in anoligonucleotide modified at either the base or sugar relatively to themonomers not modified at either the base or sugar may be less than 99%,less than 95%, less than 90%, less than 85%, less than 75%, less than70%, less than 65%, less than 60%, less than 50%, less than 45%, lessthan 40%, less than 35%, less than 30%, less than 25%, less than 20%,less than 15%, less than 10%, less than 5%, less than 1%, more than 99%,more than 95%, more than 90%, more than 85%, more than 75%, more than70%, more than 65%, more than 60%, more than 50%, more than 45%, morethan 40%, more than 35%, more than 30%, more than 25%, more than 20%,more than 15%, more than 10%, and more than 5% or more than 1%.

Lipids and/or peptides may also be conjugated to the oligonucleotides.Such conjugation may both improve bioavailability and prevent theoligonucleotide from activating RNase H and/or recruiting the RNAimachinery. Conjugation of larger bulkier moieties is typically done atthe central part of the oligonucleotide, e.g. at any of the most central5 monomers. Alternatively, at one of the bases complementary to one ofposition 1-6 of SEQ ID NO: 1 or one of position 22-27 of SEQ ID NO: 2.In yet another embodiment, the moiety may be conjugated at the 5′end orthe 3′end of the oligonucleotide. One exemplary hydrophobic moiety is acholesterol moiety that may be conjugated to the oligonucleotidepreventing the oligonucleotide from recruiting the RNAi machinery andimproving bioavailability of the oligonucleotide. For example, thecholesterol moiety may be conjugated to one or more of the nucleobasescomplementary to positions 22-27 of the sequence of SEQ ID NO: 2, at the3′end of the oligonucleotide, or at the 5′end of the oligonucleotide.

Different modifications may be placed at different positions within theoligonucleotide to prevent the oligonucleotide from activating RNase Hand/or being capable of recruiting the RNAi machinery.

In a particular embodiment, phosphorothioate internucleotide linkagesmay connect the monomers in an oligonucleotide to improve thebiostability of the oligonucleotide. All linkages of the oligonucleotidemay be phosphorothioate linkages. In another embodiment, the fraction ofphosphorothioate linkages may be less than 95%, less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 50%, more than 95%, more than 90%, more than 85%,more than 80%, more than 75%, more than 70%, more than 65%, more than60% and more than 50%.

In an embodiment, the oligonucleotide may not comprise any RNAnucleobases. This may assist in preventing the oligonucleotide frombeing capable of recruiting the RNAi machinery increasing biostabilityof the oligonucleotide. For example, the oligonucleotide may consist ofLNA and DNA nucleobases and these may be connected by phosphorothioatelinkages as outlined above. In alternative embodiments, theoligonucleotide does not comprise any DNA nucleobases. In alternativeembodiments, the oligonucleotide does not comprise any morpholino and/orLNA nucleobases.

In an embodiment, the oligonucleotide may comprise a mix of DNAnucleobases and RNA nucleobases to prevent the oligonucleotide fromactivating RNase H and prevent the oligonucleotide from recruiting theRNAi machinery. For example, DNA and RNA nucleobases may be alternatedalong the length of the oligonucleotide, or alternatively one or moreDNA nucleobases may be located adjacent one another and one or more RNAnucleobases may be located adjacent one another.

In another particular embodiment, the oligonucleotide comprises a mix ofLNA monomers and 2′-O-methyl RNA nucleobases. As above, LNA and2′-O-methyl RNA nucleobases may be alternated along the length of theoligonucleotide, or alternatively one or more LNA nucleobases may belocated adjacent one another and one or more 2′-O-methyl RNA nucleobasesmay be located adjacent one another.

In some embodiments, the number of nucleobases present in anoligonucleotide that increase the affinity of the oligonucleotide forcomplementary sequences is at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, or at least 22 nucleobases. In some embodiments, the number ofnucleobases present in a oligonucleotide that increase the affinity ofthe oligonucleotide for complementary sequences is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22nucleobases.

In particular embodiments, the nucleobases that increase the affinity ofthe oligonucleotide for complementary sequences may be located at theflanks of the oligonucleotide, i.e. at or near either or both of the 5′and 3′ ends of the oligonucleotide, or may be located at or near thecentre of the oligonucleotide. The nucleobases that increase theaffinity of the oligonucleotide for complementary sequences may also bedistributed evenly across the length of the oligonucleotide.

Table 1 sets out exemplary oligonucleotide sequences for use inaccordance with the present invention. In a particular exemplaryembodiment, the oligonucleotide has a sequence as set forth in SEQ IDNO: 5.

TABLE 1  Oligonucleotide sequences Oligonucleotide Sequence (5′-3′)¹SEQ ID NO TUCACAGUTGCUUCA 3 TUCACAGUTGCTTCA 4 T UCACAGU TGCTTCA (CD5-2)5 T U C A C A G U T G C U U CA (CD5-1) 6 T UCA C AGU TGCTTCA (CD5-3) 7¹Single underlining represents an LNA monomer; double underliningrepresents a 2′ O-methyl RNA monomer; bold represents a UNA monomer

Oligonucleotides used in the present invention may be administered inaccordance with the embodiments disclosed herein in the form ofpharmaceutical compositions, which compositions may comprise one or morepharmaceutically acceptable carriers, excipients or diluents. Suchcompositions may be administered in any convenient or suitable routesuch as by parenteral (e.g. subcutaneous, intraarterial, intravenous,intramuscular), oral (including sublingual), nasal or topical routes. Incircumstances where it is required that appropriate concentrations ofthe oligonucleotide are delivered directly to the site in the body to betreated, administration may be regional rather than systemic. Regionaladministration provides the capability of delivering very high localconcentrations of the oligonucleotide to the required site and thus issuitable for achieving the desired therapeutic or preventative effectwhilst avoiding exposure of other organs of the body to the compound andthereby potentially reducing side effects.

Oligonucleotides of the invention may be packaged and delivered insuitable delivery vehicles which may serve to target or deliver theoligonucleotides, and optionally one or more additional agents to therequired tumour site. By way of example, the delivery vehicle maycomprise liposomes, or other liposome-like compositions such as micelles(e.g. polymeric micelles), lipoprotein-based drug carriers,microparticles, nanoparticles, or dendrimers.

Liposomes may be derived from phospholipids or other lipid substances,and are formed by mono- or multi-lamellar hydrated liquid crystalsdispersed in aqueous medium. Specific examples of liposomes used inadministering or delivering a composition to target cells are DODMA,synthetic cholesterol, DSPC, PEG-cDMA, DLinDMA, or any other non-toxic,physiologically acceptable and metabolisable lipid capable of formingliposomes. The compositions in liposome form may contain stabilisers,preservatives and/or excipients. Methods for preparing liposomes arewell known in the art, for example see Methods in Cell Biology, VolumeXIV, Academic Press, New York, N.Y. (1976), p. 33 ff., the contents ofwhich are incorporated herein by reference. Biodegradable microparticlesor nanoparticles formed from, for example, polylactide (PLA),polylactide-co-glycolide (PLGA), and epsilon-caprolactone ({acute over(ε)}-caprolactone) may be used.

Other means of packaging and/or delivering oligonucleotides, andoptionally one or more additional agents, in order to facilitatedelivery to the tumour site will also be well known to those skilled inthe art. By way of example only, delivery platforms may includeRNA-lipoplex technologies comprising cationic lipids, fusogenic orstabilising co-lipids, and PEGylated lipids.

Any suitable amount or dose of an oligonucleotide of the invention maybe administered to a subject in need in accordance with the presentinvention. The therapeutically effective amount for any particularsubject may depend upon a variety of factors including: the tumour beingtreated and the severity of the tumour; the activity of the conjugateemployed; the composition employed; the age, body weight, generalhealth, sex and diet of the subject; the time of administration; theroute of administration; the rate of sequestration of the molecule oragent; the duration of the treatment; drugs used in combination orcoincidental with the treatment, together with other related factorswell known in medicine. One skilled in the art would be able, by routineexperimentation, to determine an effective, non-toxic amount of proteinconjugate to be employed.

The skilled addressee will recognise that in determining an appropriateand effective dosage range for administration to humans based on themouse studies exemplified herein, dose escalation studies would beconducted. The skilled addressee would therefore appreciate that theabove mentioned doses and dosage ranges are exemplary only based on thedoses administered in the mouse studies exemplified herein, and theactual dose or dosage range to be employed in humans may be varieddepending on the results of such dose escalation studies. Based on thedata exemplified herein, the appropriate and effective dose or dosagerange to be administered to humans can be determined by routineoptimisation, without undue burden or experimentation.

Examples of pharmaceutically acceptable carriers or diluents aredemineralised or distilled water; saline solution; vegetable based oilssuch as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil,sesame oil, arachis oil or coconut oil; silicone oils, includingpolysiloxanes, such as methyl polysiloxane, phenyl polysiloxane andmethylphenyl polysolpoxane; volatile silicones; mineral oils such asliquid paraffin, soft paraffin or squalane; cellulose derivatives suchas methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols,for example ethanol or iso-propanol; lower aralkanols; lowerpolyalkylene glycols or lower alkylene glycols, for example polyethyleneglycol, polypropylene glycol, ethylene glycol, propylene glycol,1,3-butylene glycol or glycerin; fatty acid esters such as isopropylpalmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone;agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly.Typically, the carrier or carriers will form from 10% to 99.9% by weightof the compositions.

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. The formulation must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of superfactants. Thepreventions of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilisation. Generally, dispersions are prepared byincorporating the various sterilised active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the active agents are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 μg and 2000 mg of active.

Tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: a binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavouring such as cherry or orange flavour. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theoligonucleotides may be incorporated into sustained-release preparationsand formulations.

Embodiments of the present invention also provide kits for use inaccordance with the invention. For example, kits of the invention maycontain one or more Blockmirs disclosed herein, and optionally scrambledoligonucleotides for use as controls. Such kits may be used, forexample, in medical or biological research activities, includinginvestigations into neutrophil activity, vascular permeability orinflammation. Kits according to the present invention may also includeother components required to use the Blockmirs, such as buffers and/ordiluents. The kits typically include containers for housing the variouscomponents and instructions for using the kit components in the methodsof the present invention.

Tumour Therapies

Particular embodiments disclosed herein provide for the sensitization oftumours and tumour cells to chemotherapeutic agents, immunotherapyagents or radiotherapy using oligonucleotides as disclosed herein. Thetumour or tumour cells may display resistance to the chemotherapeuticagent or immunotherapy agent in the absence of treatment with theoligonucleotide.

Thus, embodiments of the invention contemplate combination treatments,wherein administration of the oligonucleotide is in conjunction with oneor more additional anti-tumour therapies. Such additional therapies mayinclude, for example, radiotherapy, chemotherapy or immunotherapy/immunestimulation/deletion of stromal immune cells known to foster tumourgrowth, such as myeloid suppressor cells and regulatory T cells.Contemplated herein are synergistic combinations in which thecombination treatment is effective in inhibiting growth, or reducingviability, of tumour cells, to a greater extent than either component ofthe combination alone. Thus, in some embodiments a synergisticallyeffective amount of oligonucleotide and, for example, a chemotherapeuticagent or immunotherapeutic agent is administered to a subject. Asynergistically effective amount refers to an amount of each componentwhich, in combination, is effective in inhibiting growth, or reducingviability, of cancer cells, and which produces a response greater thaneither component alone.

For such combination therapies, each component of the combinationtherapy may be administered at the same time, or sequentially in anyorder, or at different times, so as to provide the desired effect.Alternatively, the components may be formulated together in a singledosage unit as a combination product. When administered separately, thecomponents may be administered by the same route of administration, ordifferent routes of administration.

Immunotherapy or immune stimulation may comprise, by way of exampleonly, adoptive cell transfer or the administration of one or moreanti-tumour or immune checkpoint antibodies, small molecules, peptides,oligonucleotides, mRNA therapeutics,bispecific/trispecific/multispecific antibodies, domain antibodies,antibody fragments thereof, other antibody-like molecules (such asnanobodies, affibodies, T and B cells, ImmTACs, Dual-AffinityRe-Targeting (DART) (antibody-like) bispecific therapeutic proteins,Anticalin (antibody-like) therapeutic proteins, Avimer (antibody-like)protein technology, anti-tumour vaccines or immune-cell modulatingreagents. Adoptive cell transfer typically comprises the recovery ofimmune cells, typically T lymphocytes from a subject and introduction ofthese cells into a subject having a tumour to be treated. The cells foradoptive cell transfer may be derived from the tumour-bearing subject tobe treated (autologous) or may be derived from a different subject(heterologous). Suitable antibodies for use in immunotherapy or immunestimulation may include anti-CTLA4 antibodies or anti-PD-1 antibodies.However these are provided by way of example only, and those skilled inthe art will appreciate that other antibodies directed to T cells orantibodies directed to other tumour cell markers may be employed. Theidentity of suitable anti-tumour antibodies will depend, for example, onthe nature or type of tumour to be treated. Suitable anti-tumourantibodies will be well known to those skilled in the art (see, forexample, Ross et al., 2003). Cells for adoptive cell transfer andanti-tumour or immune checkpoint antibodies small molecules, peptides,oligonucleotides, mRNA therapeutics,bispecific/trispecific/multispecific antibodies, domain antibodies,antibody fragments thereof, other antibody-like molecules anti-tumourvaccines or immune-cell modulating reagents may be regarded,collectively, as immunotherapy agents.

Suitable chemotherapeutic agents may be, for example, alkylating agents(such as cyclophosphamide, oxaliplatin, carboplatin, chloambucil,mechloethamine and melphalan), antimetabolites (such as methotrexate,fludarabione and folate antagonists) or alkaloids and other antitumouragents (such as vinca alkaloids, taxanes, camptothecin, doxorubicin,daunorubicin, idarubicin and mitoxantrone). In further embodimentschemotherapeutic agents may be, for example, targeted therapies, smallmolecule therapies, kinase inhibitors, including but not limited toprotein or lipid kinase inhibitors such as inhibitors of PI3 kinase, PIMfamily kinase members, receptor tyrosine kinase (RTK), Flt-3, EGFR orHER2, MEK, BRaf or an anthracyclin, a taxane, a platin, a nucleotideanalog, a hormone therapeutic agent, an anti-tumour compound that haspotential radiosensitising and/or chemosensitising effects, such aschloroquine; an mTOR inhibitor, an Akt or PI3-K inhibitor, a JAKinhibitor; an agent that modulates the DNA damage response mechanismand/or the stress signaling pathway, an inhibitor of p38 and/or NF-KB ora BCL-2 family inhibitor. However these are provided by way of exampleonly, and those skilled in the art will appreciate that otherchemotherapeutic agents may be employed.

In embodiments, the invention provides a method for modulatingnormalising tumour-associated endothelial cells, normalising tumourvasculature and/or improving vascular function in a tumour byadministration of the oligonucleotides of the invention. Thenormalization of tumour vasculature and improvement of vessel functionmay be determined, assessed or measured by a number of means orparameters well known to those skilled in the art. By way of exampleonly, normalization of tumour vasculature and improvement of vesselfunction may comprise or be characterized by one or more of: change inmorphology of endothelial cells, change in VE-cadherin expression,selective loss of large vessels; increase in number of small vessels,increased pericyte coverage of vessels, altered collagen IV coverage ofvessels, reduced vessel permeability, reduced vessel hypoxia, increasedvessel perfusion, and enhanced infiltration of immune cells. In aparticular embodiment the immune cells are lymphocytes. In furtherembodiments, lymphocytes comprise CD8+ T cells, CD4+ T cells and/or NKcells. In further embodiments, the invention provides a method forreducing tumour hypoxia.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

The present invention will now be described with reference to thefollowing specific examples, which should not be construed as in any waylimiting the scope of the invention.

EXAMPLES

The following examples are illustrative of the invention and should notbe construed as limiting in any way the general nature of the disclosureof the description throughout this specification.

General Methods Oligonucleotides

Blockmir CD5-2 was synthesized by Mirrx Therapeutics. The sequences ofoligonucleotides used in the experiments described in the followingexamples are provided in Table 2 and in the Sequence Listing appearingat the end of the specification.

TABLE 2  Oligonucleotide sequences Oligonucleotide Sequence (5′-3′)¹SEQ ID NO CD5-2 T UCACAGU TGCTTCA 5 Control Blockmir T C C A G A G A TGG T U GA 8 ¹Single underlining represents an LNA monomer; doubleunderlining represents a 2′ O-methyl RNA monomer.

RIP-TAG5 Tumour Model

RIP1-Tag5 mice (C3H background) express the oncogenic simian virus(SV40) large T antigen (Tag) under the control of the rat insulin genepromoter (RIP) in pancreatic β cells, and develop spontaneous pancreatictumours. Control Blockmir or Blockmir CD5-2 was injected systemicallyvia the tail-vein into the 27-week old RIP-Tag5 mice at a dose of 30 mgper kg of body weight in 100 μl nuclease free water. FITC-lectin wasintravenously injected into tumour-bearing mice on day 7 following theinjection of control Blockmir or Blockmir CD5-2. Fifteen minutes later,mice were perfused with PBS, tissues excised, and immunofluorescentstaining for CD31 and FITC-lectin was performed to evaluate thepercentage of perfused tumour vessels.

Adoptive Transfer of CD8+ T Cells

27-week old RIP-Tag5 mice received adoptive transfers of 2.5×10⁶ Tagspecific activated CD8+ T cells. The mice were sacrificed at day 12 andanalyzed by immunofluorescent staining for CD8+ cell infiltration.

B16F10 Melanoma Model

B16F10 melanoma cells were cultured in DMEM containing 10% FCS, 100 U/mlpenicillin, and 100 m/ml streptomycin. 4×10⁵ B16F10 melanoma cells in200 μl 1× Dulbecco's Phosphate Buffered Saline (DPBS) (LifeTechnologies) were injected subcutaneously into the dorsal right flankregion of female C57BL6 mice or into nude mice (6-8 weeks of age). Whenthe tumours became palpable, control or CD5-2, dissolved in 100 μlnuclease free water was injected systemically via the tail-vein into themice at a dose of 30 mg per kg of body weight. Tumour volumes weremeasured every day from day 5 following injection using a digitalcaliper based on the formula: V=JI×[d²×D]/6, where d is the minor tumouraxis and D is the major tumour axis. The mice were sacrificed before thetumour size reached ethical limits (1000 mm³) within 3 weeks.

Evaluation of Tumour Vascular Morphology Using Scanning ElectronMicroscope (SEM)

The tumour tissue was harvested from the mouse and rinsed in saline toremove blood and debris on the surface of the tissue. SEM fixative (2.5%SEM grade glutaraldehyde, 2% formaldehyde pH 7.4, 2 mM calcium chloride,2% sucrose and 0.1M Cac buffer pH 7.4) was then taken up into a syringeand directly injected into the tumour until it was hard (needlefixation). Care was taken to keep the injecting pressure low to avoiddestroying the blood vessels. The tumours were cut into smaller piecesover SEM fixative and incubated in SEM fixative for 72 h at 4° C.

Measurement of Tumour Vascular Permeability

To evaluate tumour vascular permeability, fluorescent 50 nm polymermicrospheres R50 (250 μl/kg) were diluted in 0.9% NaCl to a volume of100 μl and injected into the tumour-bearing C57BL6 mouse via the tailvein. The fluorescent microspheres were allowed to circulate for 6 hoursbefore the tumours were harvested, embedded in optimal cuttingtemperature (OCT) compound, and 8 μm frozen sections were cut in acryostat (Leica, Germany). Specimens were examined with a confocalfluorescence microscope (Leica SP5) and quantified with Image J software(National Institute of Mental Health, MD, USA).

Evaluation of Tumour Vascular Perfusion

To assess tumour vascular perfusion, tumour-bearing mice were injectedwith 150 μl of 2 mg/ml fluorescein isothiocyanate-conjugated tomato(Lycoper-siconesculentum) lectin (Vector Laboratories) diluted in 0.9%NaCl intravenously into the tail vein. After FITC-lectin was allowed tocirculate for 5 min, the tumours were excised, embedded in optimalcutting temperature (OCT) compound, and 8 μm frozen sections were cut ina cryostat. The frozen tumour sections were fixed, blocked, incubatedwith CD31 antibody, and then incubated with Alexa 647 goat anti-ratsecondary antibody. Specimens were examined with a confocal fluorescencemicroscope (Leica SP5) and a perfusion index was quantified as thepercentage of lectin-positive vessels per CD31-positive vessel in eachconfocal fluorescent microscopic field.

Assessment of Tumour Hypoxia

In order to measure tumour hypoxia, tumour-bearing mice were injectedintravenously with 60 mg/kg of Hypoxyprobe-1 (HP2-100; Chemicon,Temecula, Calif.) that had been resuspended at a concentration of 30mg/ml in 0.9% sterile saline. The solution was allowed to circulate for90 minutes before the tumours were removed, embedded in OCT compound,and 8 μm frozen sections were cut in a cryostat. The frozen tumoursections were fixed, blocked, and incubated with rabbit anti-CD31 andmouse anti-pimonidazole (Chemicon) primary antibodies, followed byincubation with Alexa 647 goat anti-mouse and Alexa 488 goat anti-rabbitsecondary antibodies using a mouse-on-mouse staining kit (VectorLaboratories, Burlingame, Calif.). Six random photographs were taken ofeach tissue and an average of three mice per group were used to quantifyhypoxia area.

TUNEL Staining

Cell apoptosis in OCT-embedded melanoma tissue was determined byTdT-mediated dUTP-biotin nick-end labelling (TUNEL) using in situ celldeath detection kit (Roche) following the manufacturer's instructions.After blocking with 0.5% casein for 1 h, sections were incubated for 1 hat 37° C. in the dark under humidified atmosphere with 50 μl mixture ofenzyme solution (TdT) and label solution (fluorescein-dUTP) at ratio of1:25, which was followed by washed twice with PBS (5 min each) andcounterstained with a 400 ng/ml solution of DAPI for 15 min in the dark.After two further washes in PBS, the sections were mounted under coverslip with mounting medium.

Adoptive Transfers in RIP1-Tag5 Tumour Model

RIP1-Tag5 transgenic mice were bred on a C3HeBFe background. Micetransgenic for a Tag-specific T cell Receptor (TCR), restricted toH-2K^(k) (referred to as TagTCR8) were used. Tumour-bearing RIP1-Tag5mice were treated at week 27. Adoptive transfers of in vitro activatedCD8 T cells were performed as previously described (Johansson et al.,2012). Briefly, CD8+ T cells were harvested from TagTCR8 lymph nodes andspleen, and activated for 3 days in the presence of 10 U/ml IL2(Peprotech) and 25 nM Tag peptide 560-568 (SEFLIEKRI). Tumour-bearingRIP1-Tag5 mice received a total of 2.5×10⁶ CD8+ T cells i.v. and i.p, onday 6 after miRNA injection. Tumours were analysed 4 days after adoptivetherapy for tumour infiltrating lymphocytes.

MC38 Colon Cancer Model

1×10⁶ MC38 tumour cells (in 100 μl PBS) were subcutaneously injectedinto mice on day 0. 250 μg of control Ig (2A3) and purified anti-mousePD1 mAb (RMP1-14) were intraperitoneally injected into the mice on days8, 12 and 16. 30 mg/kg control and CD5-2 were intravenously injectedinto the mice on day 8. Tumour growth was measured using a digitalcaliper and tumour size was presented as mean±SEM. The tumours wereharvested from mice that had been treated with different reagents andprocessed for flow cytometric analysis using methods well known in theart.

Monitoring Tumour Growth Using In Vivo Imaging System

B16F10-luc-G5 melanoma cells continuously expressing luciferase weremaintained in DMEM with 10% FCS, 100 U/ml penicillin, and 100 μg/mlstreptomycin at 37° C. in a humidified atmosphere of 5% CO₂. 1×10⁶B16F10-luc-G5 melanoma cells in 200 μl× DPBS (Life Technologies) wereinjected subcutaneously into the dorsal right flank region of Albino B6(C57BL/6J-Tyr<c-2J>) mice. When the tumours became palpable (generallyday 9 following the injection of B16F10-luc-G5 cells), control or CD5-2was injected systemically via the tail-vein into the mice at a dose of30 mg per kg of body weight.

Tumour growth was monitored using the Xenogen IVIS 200 imaging system(Caliper Life Sciences) and images were taken using Living ImageSoftware every 3 days once the presence of tumour was confirmed. Micewere anaesthetised during imaging process using isoflurane/oxygengaseous anaesthetic (induced at 4% isoflurane and maintained at 2%isoflurane) and given intraperitoneal injections of 200 μl D-luciferin(10 μl/g body weight of 15 mg/ml stock solution, Gold Biotechnology).Each set of Albino B6 received injections within 40 seconds and in thesame order. Images measuring the bioluminescent activity of theluciferase enzyme were acquired exactly at 15 min post intraperitonealinjections (3 min exposure, no time delay). The luminescent camera wasset to medium binning, if/stop, blocked excitation filter, and openemission filter. The photographic camera was set to medium binning and8f/stop. Field of view was set to E (22 cm) to image 5 mice at once.Identical settings were used to acquire each image and region ofinterest. Images were quantified by using LIVINGIMAGE 2.50 software.

Monitoring Metastasis Using In Vivo Imaging System

Studies of experimental pulmonary metastasis were carried out usingB16F10-luc-G5 cells that had been engineered to stably express fireflyluciferase. Cells were injected intravenously into the lateral tail veinof Albino B6 (C57BL/6J-Tyr<c-2J>) mice (10-12 weeks old). Two days priorto the injections of B16F10-luc-G5 cells, control or Blockmir CD5-2dissolved in 100 μl nuclease free water was injected via the tail-veininto the mice at a dose of 30 mg per kg of body weight. Anotherinjection of control or Blockmir CD5-2 was performed two days after thecell injection. Metastasis was monitored using the Xenogen IVIS 200imaging system (Caliper Life Sciences) and images were taken usingLiving Image Software every 3 days from day 10 following the injectionsof B16F10-luc-G5 cells. Mice were anaesthetised during imaging processusing isoflurane/oxygen gaseous anaesthetic (induced at 4% isofluraneand maintained at 2% isoflurane) and given intraperitoneal injections of200 μl D-luciferin (10 μl/g body weight of 15 mg/ml stock solution). Theparameter settings of IVIS are described in details as above mentioned.

Statistics

Statistical analyses using a two-tailed Student's t-test were performedwith Graph-Pad Prism 5.0 (GraphPad software, San Diego, Calif., USA).Data are presented as mean±standard error (S.E.M) or mean±standarddeviation (S.D.), as indicated in figure legends. Differences wereconsidered statistically significant at P<0.05.

Example 1—CD5-2 Facilitates Infiltration of CD8+ T Cells into Tumoursand Induces Tumour Apoptosis

RIP1-TAG5 mice were injected with control Blockmir or Blockmir CD5-2 at27 weeks of age. Mice were then injected with Tag specific activatedCD8+ T cells 2 days later and sacrificed a further 12 days later.Infiltration of CD8+ T cells was determined by immunofluorescentstaining for CD8. Mice injected with CD5-2 demonstrated an increasedratio of infiltrated T cells (relative to cells in the field of view)compared with those injected with control Blockmir (FIG. 1), indicatingthat Blockmir CD5-2 promotes tumour infiltration by adoptivelytransferred T cells. Thus CD5-2 can modulate the tumour microenvironmentto increase sensitivity of solid tumours to an immune response.

In a further model, the B16F10 melanoma model, infiltration ofendogenous T cells into the middle of the tumour parenchyma is increasedin Blockmir CD5-2 treated mice compared to that in the control Blockmirtreated mice (FIG. 2) demonstrating that Blockmir CD5-2 may be used tofacilitate immune therapies in tumour treatment. In particular, whenCD8+ staining was used to evaluate the infiltration of cytotoxic T cellsthere was no significant difference found in the number of infiltratedCD8+ T cells in tumour parenchyma between control and CD5-2 treatedgroups by immunofluorescence staining (FIG. 2A). However, in 8/10 miceanalysed, the CD8+ T cells in CD5-2-treated tumours were positioned morecentrally in the tumour, whereas in the control treated tumours the CD8+T cells were mainly located in the marginal area of the tumours (10/10mice) (FIG. 2B). This effect was specific for CD8+ T cells as nosignificant change in the number or localisation of CD4+ T cells, CD45+lymphocytes or in F4/80+ monocytes (data not shown) was observed.

Furthermore, CD5-2 resulted in a significant increase in apoptosiswithin the tumour mass as measured by TUNEL positive cells (FIG. 2C).

The position of the CD8+ T cells and the enhanced degree of apoptosisfollowing CD5-2 treatment suggests that the retardation of tumour growthis likely to be immune mediated. To confirm this possibility theinventors used immunocompromised nude mice. CD5-2 had no effect on thetumour growth (FIG. 2D) in nude mice. However, it did alter thevasculature in these mice as both pericyte (FIG. 2E) and smooth musclecell (FIG. 2F) coverage were enhanced following the delivery of CD5-2.

Example 2—Blockmir CD5-2 Modulates Tumour Vasculature and TumourVascular Cell Morphology

Tumour vasculature in mice with B16F10 melanoma was visualised usingimmunohistochemistry for CD31. Mice treated with Blockmir CD5-2 6 to 8days prior to sacrifice displayed smaller vessels within tumours thancontrol Blockmir treated mice. While the number of vessels per field inBlockmir CD5-2 treated mice was higher than that of control Blockmirtreated mice, the average blood vessel volume was significantly reducedin mice that received Blockmir CD5-2 (FIG. 3), indicating that BlockmirCD5-2 can alter the morphology of tumour vasculature without vesselpruning. Consistent with this, CD5-2 treated ECs in vitro showed nochanges in angiogenic-associated characteristics including proliferation(data not shown), senescence (data not shown) and migration (data notshown).

FIG. 4 demonstrates scanning electron microscopy of blood vessels withina B16F10 melanoma. Abnormal morphology of endothelial cells is seen incontrol Blockmir treated mice in which endothelial cells displayproperties of a non-quiescent, hyperactive endothelium appearing looselyconnected and detached from each other. Endothelial cells are multilayered, rounded, disconnected and display luminal protrusions. Obviousgaps in the vessel wall are present, showing weak characteristics ofcell-cell contact. Treatment with Blockmir CD5-2 normalises endothelialcell appearance of the tumour vessels as evidenced by increasedorganisation into a flattened single monolayer with cobblestoneappearance indicative of a quiescent and less active endothelium.

Pericyte coverage of the endothelium is also altered by Blockmir CD5-2treatment. Pericytes are characteristically poorly attached to tumourvasculature. In the B16F10 model of melanoma, mice treated with BlockmirCD5-2 demonstrate significantly higher colocalisation of pericytes andendothelial cells than mice treated with control Blockmir (FIGS. 5A and5B) indicating Blockmir CD5-2 helps to normalise tumour vasculature.Smooth muscle cell coverage, defined by αSMA expression, is alsoincreased in tumour-associated vasculature following CD5-2administration (FIGS. 5C and 5D).

Blockmir CD5-2 also regulates expression of VE-cadherin intumour-associated vessels, VE-cadherin being increased in thevasculature of Blockmir CD5-2 treated mice relative to control Blockmirtreated mice (FIG. 6). Similarly, Blockmir CD5-2 treatment increasedcollagen IV coverage of tumour vessels compared to control treatedanimals (FIG. 7) implicating an effect of Blockmir CD5-2 on theextracellular matrix and the integrity of the basement membrane.Together, these structural changes suggest that tumour-associatedvessels are “normalised” by CD5-2 treatment.

Example 3—Tumour Vasculature Function is Altered by Blockmir CD5-2

In addition to altered structure of tumour vasculature, Blockmir CD5-2modulated permeability and perfusion of tumour vasculature in the B16F10mouse melanoma model. FIG. 8 demonstrates the extravasion of R50fluorescent microspheres injected into control Blockmir or BlockmirCD5-2 treated mice. CD5-2 reduced the vessel permeability as measured bythe number of R50 fluorescent microspheres within the tumour parenchyma(FIG. 8A). As shown in FIG. 8B, there was a significant reduction in theratio of microspheres to endothelial marker (CD31) in Blockmir CD5-2treated animals relative to controls, indicating reduced leakiness ofvessels with Blockmir CD5-2 treatment. Consistent with this decreasedvascular permeability there was a decrease in the extent of fibrinogendeposited into the matrix (FIGS. 8C and 8D).

Furthermore, the percentage of perfused tumour vessels, as demonstratedwith FITC-lectin/CD31 immunofluorescence colocalisation, was increasedwith Blockmir CD5-2 administration relative to control Blockmirtreatment (FIG. 9). The hypoxic microenvironment of a tumour is chieflyrelated to an abnormal vessel network and the consequently abnormalperfusion. A reduction in hypoxic area of the tumour, as detected bypimonidazole staining of the hypoxia probe Hypoxyprobe-1, was alsodemonstrated Blockmir CD5-2 treated mice (FIG. 10) demonstrating afunctional change in tumour vasculature in response to Blockmir CD5-2.

In the RIP-TAG5 mouse model, detection of FITC-lectin immunofluorescencewas also increased relative to CD31 in mice treated with CD5-2 relativeto those treated with a control Blockmir (FIG. 11) indicating improvedperfusion of the tumour vasculature in the presence of CD5-2.

Example 4—CD5-2 Enhances Immunotherapeutic Effects

The effects of CD5-2 to normalise the vasculature of thetumour-associated vessels and the enhancement of CD8⁺ T cells into thecentral regions of the microenvironment suggest that it might functionto promote immunotherapy. To test this the inventors used two models,the RIP-Tag5 pancreatic neuroendocrine tumour model as a model foradaptive T cell therapy and the colon carcinoma MC38 model, which issensitive to check-point blockade (the administration of anti-PD1antibody).

In the RIP-Tag5 model, CD5-2 enhanced the perfusion of the vessels,confirming an effect on the vasculature, similar to that seen in theB16F10 model (FIG. 12A). Tumour-specific CD8⁺ T cells were activated exvivo and these cells were then adoptively transferred intotumour-bearing RIP-Tag mice that had previously been treated with eitherCD5-2 or control. CD5-2 treatment resulted in a significant enhancementin the infiltration of the activated tumour specific CD8+ T cells (FIG.12B).

In the MC38 model, a single delivery of CD5-2 resulted in a significantinhibition of tumour growth, to similar levels as for anti-PD-1 alone(FIG. 12C). Strikingly, the combination treatment of 3 injections ofanti-PD-1 and a single injection of CD5-2 resulted in a significantinhibition of tumour growth, over that seen for either treatment alone.Analysis of the immune infiltrate showed that CD5-2+ anti-PD-1 treatmentresulted in a significant increase in the CD8+/CD11b/Gr1 hi ratio (FIG.12D). Further, the percentage of CD8+ T cells that are Granzyme Bpositive, as an indication of activation, was increased by CD5-2 byitself and also by anti-PD-1 (FIG. 12E). CD5-2 treatment of ECmonolayers in vitro results in a decrease in the PD-1 ligand, PDL1 (FIG.12F), which may contribute to an increase in activation of CD8+ T cellsin an in vivo context.

Example 5—Blockmir CD5-2 Reduces Tumour Metastasis

B16F10 melanoma cells expressing luciferase were injected into C57BL/6mice and visualised with the injection of luciferin at 12, 15 or 18 dayspost-tumour cell injection. In mice treated with Blockmir CD5-2,bioluminescence was qualitatively reduced compared to mice injected withcontrol Blockmir (FIG. 13). FIG. 12D demonstrates a non-significantreduction in bioluminescence at 18 days post-cell injection in micetreated with Blockmir CD5-2 relative to control Blockmir. Additionally,the lungs and livers harvested from Blockmir CD5-2 treated mice aftersacrifice demonstrated qualitatively less bioluminescence than did thosefrom control Blockmir treated mice (FIG. 14) indicating that BlockmirCD5-2 can reduce metastasis.

1. A method for increasing the sensitivity of a tumour to immunotherapy,chemotherapy or radiotherapy, wherein the method comprises administeringto a subject in need thereof an effective amount of an oligonucleotidecomprising a contiguous sequence complementary to at least 8 contiguousbases of an RNA sequence comprising SEQ ID NO: 1, or SEQ ID NO: 1comprising 1, 2 or 3 substitutions, wherein the oligonucleotide inhibitsthe binding of miR-27a, a variant thereof or a miRNA comprising a seedregion comprising the sequence UCACAG, to said RNA.
 2. The method ofclaim 1, wherein the method further comprises immunotherapy,chemotherapy or radiotherapy of the tumour in the subject.
 3. The methodof claim 2, wherein the oligonucleotide is administered to the subjectprior to, concomitantly with, after, or otherwise in combination with,immunotherapy, chemotherapy or radiotherapy of the tumour.
 4. The methodof any one of claims 1 to 3, wherein the oligonucleotide comprises acontiguous sequence complementary to at least 8 contiguous bases of anRNA sequence comprising SEQ ID NO: 2, or SEQ ID NO: 2 comprising 1, 2 or3 substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.
 5. The method of any one ofclaims 1 to 4, wherein the miR-27a miRNA is hsa-miR-27a comprising thenucleotide sequence set forth in SEQ ID NO:9.
 6. The method of any oneof claims 1 to 5, wherein the oligonucleotide comprises a contiguoussequence complementary to a sequence of at least or about 7 bases, atleast or about 8 bases, at least or about 9 bases, at leak or about 10bases, at least or about 11 bases, at least or about 12 bases, at leastor about 13 bases, at least or about 14 bases, at least or about 15bases, at least or about 16 bases, at least or about 17 bases, at leastor about 18 bases, at least or about 19 bases, at least or about 20bases, at least or about 22 bases, at least or about 25 bases, at leastor about 30 bases, or at least or about 35 bases of SEQ ID NO: 2, or SEQID NO: 2 comprising 1, 2 or 3 substitutions.
 7. The method of any one ofclaims 1 to 6, wherein the oligonucleotide binds to positions 22-27 ofSEQ ID NO:
 2. 8. The method of any of claims 1 to 7, wherein basepairing between the oligonucleotide and SEQ ID NO: 2 includes positions8-28, 8-27, 9-27, 10-27, 11-27, 12-27, 13-27, 14-27, 15-27, 16-27,17-27, 18-27, 19-27, 20-27, 21-27, 9-28, 10-28, 11-28, 12-28, 13-28,14-28, 15-28, 16-28, 17-28, 18-28, 19-28, 20-28 or 21-28 of SEQ ID NO:2.
 9. The method of any one of claims 1 to 8, wherein theoligonucleotide comprises the sequence set forth in SEQ ID NO: 3 or SEQID NO:
 4. 10. The method of any one of claims 1 to 9, wherein theoligonucleotide comprises one or more modified nucleobases.
 11. Themethod of claim 10, wherein the modified nucleobase is an LNAnucleobase, a UNA nucleobase or a 2′ O-methyl nucleobase.
 12. The methodof any one of claims 1 to 11, wherein the oligonucleotide comprises asequence set forth in SEQ ID NO:
 5. 13. The method of any one of claims1 to 12, wherein the immunotherapy comprises immune stimulation.
 14. Themethod of claim 13, wherein the immune stimulation comprises adoptivecell transfer or the administration of one or more anti-tumour or immunecheckpoint antibodies, small molecules, peptides, oligonucleotides mRNAtherapeutics, bispecific/trispecific/multispecific antibodies, domainantibodies, antibody fragments thereof, antibody-like molecules,anti-tumour vaccines or other immune cell modulating agents.
 15. Themethod of claim 14, wherein said adoptive cell transfer comprises thetransfer of autologous tumour infiltrating lymphocytes.
 16. The methodof claim 14, wherein the anti-tumour antibodies comprise anti-PD-1antibodies.
 17. A method for modulating tumour metastasis, the methodcomprising exposing a tumour to an effective amount of anoligonucleotide comprising a contiguous sequence complementary to atleast 8 contiguous bases of an RNA sequence comprising SEQ ID NO: 1, orSEQ ID NO: 1 comprising 1, 2 or 3 substitutions, wherein theoligonucleotide inhibits the binding of miR-27a, a variant thereof or amiRNA comprising a seed region comprising the sequence UCACAG, to saidRNA.
 18. The method of claim 17, wherein said modulating tumourmetastasis comprises reducing tumour metastasis.
 19. A method fornormalising tumour vasculature and/or improving vascular function in atumour, the method comprising exposing a tumour to an effective amountof an oligonucleotide comprising a contiguous sequence complementary toat least 8 contiguous bases of an RNA sequence comprising SEQ ID NO: 1,or SEQ ID NO: 1 comprising 1, 2 or 3 substitutions, wherein theoligonucleotide inhibits the binding of miR-27a, a variant thereof or amiRNA comprising a seed region comprising the sequence UCACAG, to saidRNA.
 20. The method of claim 19, wherein normalizing the tumourvasculature and/or improving vessel function comprises or ischaracterized by one or more of: change in morphology of endothelialcells, change in VE-cadherin expression, selective loss of largevessels; increase in number of small vessels, increased pericytecoverage of vessels, altered collagen IV coverage of vessels, reducedvessel permeability, reduced vessel hypoxia, increased vessel perfusion,and enhanced infiltration of immune cells.
 21. The method of claim 20,wherein the immune cells comprise lymphocytes, neutrophils, monocytesand/or macrophages.
 22. The method of claim 21, wherein lymphocytescomprise CD8+ T cells, CD4+ T cells and/or NK cells.
 23. A method forreducing tumour hypoxia, the method comprising exposing a tumour to aneffective amount of an oligonucleotide comprising a contiguous sequencecomplementary to at least 8 contiguous bases of an RNA sequencecomprising SEQ ID NO: 1, or SEQ ID NO: 1 comprising 1, 2 or 3substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.
 24. A method for promotingcell death of tumour cells, the method comprising exposing a tumour toan effective amount of an oligonucleotide comprising a contiguoussequence complementary to at least 8 contiguous bases of an RNA sequencecomprising SEQ ID NO: 1, or SEQ ID NO: 1 comprising 1, 2 or 3substitutions, wherein the oligonucleotide inhibits the binding ofmiR-27a, a variant thereof or a miRNA comprising a seed regioncomprising the sequence UCACAG, to said RNA.
 25. Use of anoligonucleotide comprising a contiguous sequence complementary to atleast 8 contiguous bases of an RNA sequence comprising SEQ ID NO: 1, orSEQ ID NO: 1 comprising 1, 2 or 3 substitutions, wherein theoligonucleotide inhibits the binding of miR-27a, a variant thereof or amiRNA comprising a seed region comprising the sequence UCACAG, to saidRNA for the manufacture of a medicament for sensitising tumours,modulating tumour metastasis, normalising tumour vasculature and/orpromoting cell death of tumour cells.